Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi

The globally distributed marine alga Emiliania huxleyi has cooling effect on the Earth’s climate. The population density of E. huxleyi is restricted by Nucleocytoviricota viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here, we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane. EhV-201 virions infect E. huxleyi by using fivefold vertices to bind to and fuse the virus’ inner membrane with the cell plasma membrane. Progeny virions assemble in the cytoplasm at the surface of endoplasmic reticulum–derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. The genome-filled capsids acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which enables the continuous release of virions by exocytosis.

). Structural similarity search using DALI (87) determined that it is a common structural module in many viral penton insertion domains (88).The domains D3 and D5 share similarity with insertion modules of other virus proteins that have structural functions (Table S2) (89,90).Please note that the cell envelope, which covers most E. huxleyi cells when imaged using cryo-electron microscopy (Fig. 3), is not resolved in the resin-embedded samples, probably because it was dissolved during the sample preparation procedure or not stained by osmium tetroxide and uranyl acetate used for sample contrasting.Scale bar 500 nm.

Fig. S2 .
Fig. S2.Surface layers of EhV-201 virion.(A) Plot of average pixel intensities measured along lines perpendicular to particle surface in reference-free two-dimensional class average of oblique segments of EhV-201 particle surface.Layers representing the inner membrane, capsid, and outer membrane are indicated by colored backgrounds (IM inner membrane is shown in blue, C capsid in magenta, and OM outer membrane in orange).Numbers indicate layer thickness.The outer leaflet of the outer membrane, indicated by dark orange, has a stronger density than the inner leaflet.The coloring scheme corresponds to that in Fig. 1B, C. The 95% confidence interval is indicated by grey shading.The average background intensity level is indicated by a horizontal dashed line.(B) Extended plot including region used for determination of average background value calculated from hatched area.N = 18.

Fig. S3 .
Fig. S3.Plots of Fourier shell correlation (FSC) of reconstructions of independent halves of cryo-ET and cryo-EM datasets of EhV-201 virion vertices.(A,B) Sub-tomogram reconstructions of EhV-201 virion vertex with masks limiting the size of the reconstruction to 120 nm (A) and 50 nm (B).(C) Subtomogram reconstructions of EhV-201 virion vertex with mask limiting the size of the reconstruction to 50 nm and removing the outer and inner membrane.(D) Single-particle reconstruction of EhV-201 virion vertex with masks limiting the size of the reconstruction to 50 nm.Dashed lines indicate FSC values 0.5 and 0.143.

Fig. S4 .
Fig. S4.Outer membrane of EhV-201 is decorated with transmembrane proteins.(A) Surface representation of sub-tomogram of EhV-201 vertex reconstructed using a mask with the diameter of 50 nm showing central vertex proteins (red), peripheral vertex proteins (light blue), and dimers of ridge proteins (yellow).Scale bar 10 nm.(B-G) Surface representations of transmembrane proteins in side (B,E) and bottom views (C,D,F,G).Central and peripheral vertex proteins (B-D) with underlying monomer of the penton protein in cartoon representation (purple) (B,C) and without it (D) -the cavity, where the penton protein binds is visible.Ridge protein dimer (E-G) with underlying major capsid protein capsomers in cartoon representation (blue) (E,F) and without the major capsid proteins (G).Scale bar (B-G) 5 nm.

Fig. S5 .
Fig. S5.EhV-201 capsid is organized with T = 169 quasi-symmetry.Surface representation of three cryo-ET reconstructions of angular vertices (mask diameter 120 nm) placed back into tomogram of EhV-201 virion based on coordinates obtained by three-dimensional refinement.Positions of fivefold symmetry axes are indicated by red pentagons, h and k directions are indicated by red dots.Selected penta-symmetrons are highlighted in yellow and tri-symmetron in red.Positions of selected fivefold symmetry axes are indicated by black pentagons, threefold axis by a triangle, and twofold axes by ovals.Scale bar 5 nm.

Fig. S6 .
Fig. S6.Structure of EhV-201 major capsid protein and penton protein.(A) Cartoon representation of AlphaFold2 (36) predicted structure of a monomer of EhV-201 major capsid protein.The domains J1 and J2 are colored in dark and light blue, respectively.Each domain contains two four-stranded bsheets with the b-strands conventionally named BIDG and CHEF.Domain J1 contains an insertion between b-strands D and E, DE loop, (grey) which contains amphipathic helices a3 (orange) and a4 (magenta).(B,C) Side (B) and top (C) view of capsomer formed by three monomers of the major capsid proteins shown in red, green, and blue.(D-F) Cartoon representation of EhV-201 penton protein.(D) A monomer of penton protein with single jelly roll fold domain J1 is shown in blue.The domain is formed by two four-stranded b-sheets with the b-strands named BIDG and CHEF.An insertion in the domain J1 forms domains D1-5, which are distinguished by colors (TableS2).(E, F) Pentamer of penton proteins, each of which is shown in distinct color.(G-I) Electrostatic surface potential plot of the penton protein pentamer.The surface of the penton exposed to the outside of the capsid is positively

Fig. S7 .
Fig. S7.Mass spectrometry identification of the penton protein.(A) A silver-stained SDS-PAGE gel of EhV-201 virion proteins.The band indicated by a white arrowhead contained major capsid protein.The band indicated by a black arrowhead contained the penton protein.(B) List of peptides from the penton protein that were identified using mass spectrometry analysis in the band indicated by the white arrowhead in panel A.

Fig. S8 .
Fig. S8.Inner membrane is less resolved than outer one in EhV-201 virion vertex sub-tomogram reconstruction.(A) Central sections of sub-tomogram reconstructions of vertices from EhV-201 virion with reconstruction diameter set to 120 nm (grey) and 50 nm (inset in green).No feature-based masks were applied in the reconstruction process.Scale bar 10 nm.(B) Plot of average voxel intensities measured along lines perpendicular to EhV-201 virion surface.Layers representing the inner membrane, capsid, and outer membrane are marked by colored backgrounds (IM inner membrane in blue, C capsid in magenta, and OM outer membrane in orange).The coloring scheme corresponds to that in Fig. 1B,C.The 95% confidence interval (N = 14) is indicated by grey shading.

Fig. S9 .
Fig. S9.DE loop of J1 domain of EhV-201 major capsid protein contains amphipathic helices a3 and a4.(A) Helical wheel representation of helix a3 (residues 111-123) from J1 domain of EhV-201 major capsid protein, prepared using HeliQuest server (91), indicating its amphipathic properties.Amino acids with hydrophobic side chains are shown in yellow and hydrophilic amino acids in white.(B) HeliQuest plot of helix a4 (residues 133-152).The arrows in panels (B) and (C) indicate the magnitude and direction of the hydrophobic moment.(CD) Surface representation of amphipathic helices a3 (C) and a4 (D) colored by hydrophobicity showing clustering of hydrophobic residues (yellow) on one face (top panel) whereas polar residues prevail on the opposite side (bottom panel).

Fig. S10 .
Fig. S10.Some vertices of EhV-201 virions are decorated with flexible fibers.(A-C) Projection images of 16-nm-thick sections of cryo-tomograms of EhV-201 virions.Fibers attached to some of the virion vertices are indicated by black arrowheads.Scale bar 50 nm.

Fig. S11 .
Fig. S11.Productive and abortive genome delivery of EhV-201.Scanning electron micrographs of a high-pressure vitrified and resin-embedded sample of E. huxleyi cells infected by EhV-201 at MOI = 10, 30 min post-infection.(A-E) Productive infection pathway.Genome-containing (white arrowhead) (A-E) and empty (black arrowhead) (E) EhV-201 particles attached to PM plasma membrane.(F-I)Abortive infection.Genome-containing (F) and empty (F-I) EhV-201 particles attached to SM surface membrane of E. huxleyi cells Please note that the cell envelope, which covers most E. huxleyi cells when imaged using cryo-electron microscopy (Fig.3), is not resolved in the resin-embedded samples, probably because it was dissolved during the sample preparation procedure or not stained by osmium tetroxide and uranyl acetate used for sample contrasting.Scale bar 200 nm.

Fig. S12 .
Fig. S12.Properties of EhV-201.(A) The efficiency of EhV-201 propagation on E. huxleyi strain CCMP 2090.Dot plot showing the number of plaque forming units per milliliter obtained for EhV-86 and EhV-201 propagated on E. huxleyi CCMP 2090.Mean and standard deviation (error bars) are indicated.N = 3. (B) Lysis of E. huxleyi culture by EhV-201 at various MOI.Growth curves of E. huxleyi CCMP 2090 infected by EhV-201 at MOI 0 (mock), 0.01, 1, and 10.Curves represent the 3rd-order polynomial fit to the data.Error bars correspond to the standard deviation (N = 3).(C) EhV-201 infectivity is not affected by DAPI fluorescence staining.Dot plot of the number of plaque-forming units in 100 µl of a viral lysate with and without DAPI treatment.The mean and standard deviation (error bars) are indicated.The seawater medium-treated group was used as a control (Mock).(D) Size distribution of EhV-201 assembly intermediates.The maximum outer diameters of genome packaging intermediates, full capsids, and virions were measured from cryo-tomograms of infected cells.Violin plots showing both kernel density and box plot: central black line -median; box -interquartile range; whiskers -1st and 4th data quartile without outliers; the outlier greater than 1.5 times the interquartile range is depicted by a black dot.N = 25.

Fig. S13 .
Fig. S13.Surface layers of E. huxleyi cells.(A-C) Projection images of 30-nm-thick tomogram sections of a cell from the non-calcifying E. huxleyi strain CCMP 2090, which spontaneously resumed coccoliths production.The cell is surrounded by a large number of EhV-201 virions (Vi) as it was infected at MOI = 100 and imaged at 30 min post-infection.Co coccolith, SM surface membrane, CE cell envelope, CL cytoplasmic leaflet, and PM plasma membrane.The opening in the surface membrane, cell envelope, and cytoplasmic leaflets is indicated by white arrowheads.(B,C) Virions of EhV-201 can diffuse beneath the E. huxleyi coccoliths shell as indicated by black arrowheads.The particles have diameter 209 nm SD 4.3 nm, indicating that they are virions containing outer membrane (N = 19).Scale bar 500 nm.

Fig. S14 .
Fig. S14.Morphology of native E. huxleyi cell.Scanning electron micrograph of a high-pressure vitrified and resin-embedded sample of non-calcifying E. huxleyi CCMP 2090 cell.Ch chloroplast, M mitochondrion, N nucleus, SM surface membrane, and PM plasma membrane.Please note that the cell envelope, which covers most E. huxleyi cells when imaged using cryo-electron microscopy (Fig.3), is not resolved in the resin-embedded samples, probably because it was dissolved during the sample preparation procedure or not stained by osmium tetroxide and uranyl acetate used for sample contrasting.Scale bar 500 nm.

Fig. S15 .
Fig. S15.Heterogeneity in the thickness of E. huxleyi cell envelope.A projection image of a 30-nmthick tomogram section of a control non-infected E. huxleyi cells with a thick cell envelope (CE)bottom cell, and a thin cell envelope -upper cell, surface membrane (SM), Ch chloroplast, CL cytoplasmic leaflet, N nucleus, PM plasma membrane.Scale bar 200 nm.

Fig. S16 .
Fig. S16.EhV-201 attachment to E. huxleyi cells.(A, B) Maximum intensity projections of 2.8-µm-thick volumes of fluorescence confocal sections showing plasma membrane of E. huxleyi cells in green (stained with FM 1-43) and EhV-201 in blue (stained with DAPI).(A) E. huxleyi cells infected at MOI 100.The EhV-201 particle attached to the cell surface is indicated by a white arrowhead.The inset shows details of the cell with a virus attached from the outside.(B) Non-infected control cells.Many E. huxleyi cells contain pigment granules that produce a blue signal (indicated by a black arrowhead with a white outline).However, the pigment granules were never observed to be attached to a cell surface.The inset shows detail of the fluorescent granule inside a control cell.Scale bar 5 µm.

Fig. S17 .
Fig. S17.Lysis of infected E. huxleyi cells results in release of EhV-201 virions inside vesicles.(A-D) Projection images of 30-nm-thick tomogram sections of vesicles released from a lysed E. huxleyi cell.Scale bar 200 nm.